In vitro protein tyrosine phosphatase 1B inhibition and antioxidant property of different onion peel cultivars: A comparative study

Abstract The aim of the present study was a comparative investigation of water and 70% ethanol extracts derived from yellow and red onion (Allium cepa L.) peels against diabetes and diabetic complications. The total phenolic contents (TPCs) and total flavonoid contents (TFCs) of each cultivar, measured to assess phytochemical characteristics, showed a direct correlation with the in vitro antioxidant effects. Among the two captives, the yellow onion peel extract showed higher antioxidant activity than red one. However, all extracts exhibited significant protein tyrosine phosphatase 1B (PTP1B) inhibitory activity (IC50; 0.30–0.86 μg/ml), showing water extracts more potent (IC50; approximately 0.3 μg/mL), than the 70% ethanol extracts (IC50; approximately 0.8 μg/ml). Similarly, in insulin‐resistant HepG2 cells, all extracts enhanced the glucose uptake and reduced the expression of PTP1B in a concentration‐dependent manner, water extract displaying better activity. Our results overall suggest that in vitro antioxidant and antidiabetic potentials vary among red and yellow cultivars and extracting solvents, which could therefore be a promising strategy to prevent diabetes and associated complications.


| INTRODUC TI ON
Diabetes mellitus (DM) is the most common form of metabolic disorder that damages our various organs such as heart, kidneys, blood vessels, nerves, and eyes, leading to lifelong disability and premature death. Insulin resistance with an inadequate insulin secretory response is the etiology of type 2 diabetes mellitus (T2DM; Umar, Ahmed, Muhammad, Dogarai, & Soad, 2010). It is considered as one of the most attentive chronic diseases of the recent time due to its high prevalence and significant social and economic consequences. It is predicted that more than 415 million people are suffering from diabetes in 2015. Among them, approximately 90% of the people are diagnosed with T2DM (Sun et al., 2017).
Extended research on diabetes has discovered many synthetic drugs against diabetes. Though developed therapies are able to reverse health issues/complications related to diabetes, they lead to various side effects. For many years in traditional folk medicine, diabetes and other fatal diseases have been treated orally with the variety of plant extracts. Till date, more than 1,200 plant species with antidiabetic properties have been reported (Habeck, 2003;Said et al., 2008). Nowadays, metformin is the most popular drug for DM, which was discovered with reference to biguanide compound isolated from French lilac (Oubre, Carlson, King, & Reaven, 1997). The selected plant could also be a potential candidate for this aim.
Onion (Allium cepa L.), which is consumed fresh as well as processed, is one of the most important vegetables worldwide. It belongs to the Alliaceae family and is biennial. It is commercially produced as an annual vegetable. It may differ greatly in color of outer scales (yellow, red, and white) and bulb shape (Slimestad, Fossen, & Vågen, 2007). Many studies suggest that regular consumption of onion helps to decrease the risk of several abnormalities such as neurodegenerative disorder, cancer, cataract formation, ulcer development, osteoporosis, and cardiovascular diseases (Singh et al., 2009). Onion contains various biologically active molecules such as phenolic acids, flavonoids, cepaenes, thiosulfinates, and anthocyanins (Goldman, Kopelberg, Debaene, & Schwartz, 1996). Further, flavonoids have shown other biological activity such as inhibition of plasma aggregation and cyclooxygenase (COX) activity; histamine release and slow-reacting substance of anaphylaxis (SRS-A) inhibition; and antibacterial, antiviral, anti-inflammatory, and anti allergic effects (Hope, Welton, Fiedler-Nagy, Batula-Bernardo, & Coffey, 1983). There have been various studies regarding the onion having the high level of flavonols (Hertog, Feskens, Kromhout, Hollman, & Katan, 1993;Suh, Lee, Cho, Kim, & Chung, 1999). But unfortunately, onion peel is considered as waste and more than 500,000 tons of onion waste is produced annually in the European Union alone (Benítez et al., 2011). It includes skin, outer layers, roots, and stalks. Due to its aroma and rapid development of phytopathogenic agents, it cannot be used as fodder as well as organic fertilizer. So they are dumped. Therefore, a possible solution could be the use of waste as a source of food ingredients as onion skin contains a significant amount of flavonoids than the edible portion by about 2-10 g/kg (Suh et al., 1999). In a study conducted to evaluate the anti diabetic effect of onion peel extract (Jung, Lim, Moon, Kim, & Kwon, 2011), 60% ethanol extract of onion peel ameliorated hyperglycemia and insulin resistance in high-fat diet/streptozotocin-induced diabetic rats via alleviating metabolic dysregulation of free fatty acids, suppressing oxidative stress, and upregulating peripheral glucose uptake. Similarly, a study by Lee et al. (2008) suggested that onion skin is effective in controlling hyperglycemia via αglucosidase inhibition. In addition, ethanol extract of onion peel improved exaggerated postprandial spikes in blood glucose and glucose homeostasis by inhibiting intestinal sucrase and thus delaying carbohydrate absorption (Kim, Jo, Kwon, & Hwang, 2011).
Though ample of studies concluded the antidiabetic potentials of onion peel extract in vitro and in vivo, there are limited papers on comparative study on different onion cultivars. The composition of onion varies with cultivar, stages of maturation, environment, agronomic conditions, storage time, and bulb part (Abayomi & Terry, 2009;Downes, Chope, & Terry, 2010). So it is essential to investigate the anti diabetic and antioxidant activity of onion peel (cultivars) to include as a possible food ingredient.

| Preparation of samples
The peels of onion were dried under shade and coarsely powdered for extraction. Dried red onion peel (110 g) and dried yellow onion peel (70 g) were extracted with 5 L of 70% ethanol (EtOH) for 3 times to get 15.93 g of red onion peel 70% ethanol extract (RE) and 6.24 g of yellow onion peel 70% ethanol extract (YE), respectively. Similarly, dried red onion peel (110 g) and dried yellow onion peel (70 g) were extracted with 5 L of water for 3 times to get 23.25 g of red onion peel water extract (RW) and 7.41 g of yellow onion peel water extract (YW).

| Determination of total phenolic content
The total phenolic content (TPC) of each of 70% ethanol and water extracts of dried peel of red and yellow onion was determined using the Folin-Ciocalteu reagent as described previously (Iqbal & Bhanger, 2006). The results were recorded as mg of gallic acid equivalent (GAE) per g of extract.
The GAE values were expressed as mean ± SEM of triplicate experiments.

| Determination of total flavonoid content
The total flavonoid content (TFC) of samples was measured by the aluminum chloride colorimetric method as described previously (Iqbal & Bhanger, 2006).

| Protein tyrosine phosphate 1B inhibitory assay
Protein tyrosine phosphate 1B (PTP1B) inhibitory assay of the samples was determined using method as mentioned previously . The PTP1B inhibitory activity of each sample was expressed in terms of IC 50 (μg/ml) and expressed as mean ± SEM of triplicate experiments.
α-Glucosidase inhibitory activity of each sample was expressed in terms of IC 50 (μg/ml) and expressed as mean ± SEM of triplicate experiments.

| Advanced glycation end product formation inhibitory assay
Advanced glycation end products (AGEs) formation inhibitory assay of different samples was determined as described earlier (Shrestha et al., 2018).

| Cell culture, MTT assay, and insulin resistance induction
Human hepatocarcinoma (HepG2) cells were purchased from the American Type Culture Collection (HB-8065; Manassas, VA). Cells were maintained at 37°C in a humidified atmosphere with 5% CO 2 in 10% FBS MEM. Cytotoxicity of extracts was evaluated using the MTT assay (Mosmann, 1983). For developing insulin-resistant HepG2 cell model, method by Liu et al. was followed (Liu et al., 2015). Rest of the experimental conditions and procedures were similar to those reported in our previous paper (Bhakta et al., 2017).
Experimental conditions and steps followed to evaluate glucose uptake were same as previously described .
Rosiglitazone (10 μmol/L) was used as a reference drug.

| Preparation of cell lysates and western blot analysis
Standard protocol was followed to prepare lysates of insulinresistant HepG2 cells using sample buffer and PMSF. Fifty micrograms of protein, once quantified by modified Bradford protein assay kit, was separated using a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA). The polyvinylidene difluoride (PVDF) membranes were incubated overnight on a shaker at 4°C with primary antibody prepared in 5% skim milk and visualized on X-ray film after incubating PVDF membranes with secondary antibody for 2 hr at room temperature. Band intensities were quantitated using CS analyzer software (Atto Corp., Tokyo, Japan).

| Statistical analysis
The results are presented as the mean ± standard error of the mean (SEM) following one-way ANOVA and Duncan's test (Systat Inc., Evanston, IL). A p-value <0.05 was considered significant for the differences.

| Yield (%), total phenolic content, and total flavonoid content of onion extracts
Red and yellow onions were extracted with both 70% EtOH and water to obtain respective extracts. As shown in Table 1

| 1,1-Diphenyl-2-picrylhydrazyl radical scavenging activity of onion extracts
To evaluate the antioxidant ability of onion peel of red and yellow cultivars, the 70% EtOH and water extracts were tested for in vitro 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity. Based on the formation of the DPPH-H nonradical form in the presence of hydrogen-donating antioxidants in the extracts, the DPPH radical scavenging activity was determined.
The DPPH radical scavenging activity of 70% EtOH and water extracts of red and yellow onion was tested at different concentrations using L-ascorbic acid as positive standard. The results Yield (%): The yield (w/w) percentage of the 70% EtOH and water extracts from two onion cultivars. *Significant difference between red and yellow onions (p < 0.05). **Significant difference between 70% EtOH and water extracts of onions (p < 0.05).

Extracts Yield (%) a
TA B L E 1 Total phenolic and flavonoid contents of onion extracts (mean ± SEM, n = 3) TA B L E 2 Antioxidant and antidiabetic activity of the 70% ethanol and water extracts from onion (mean ± SEM, n = 3) are demonstrated in Table 2 and Figure 1a. The extracts at a concentration of 6.4 μg/ml showed a range of 42%-66% DPPH radical scavenging activity in the concentration-dependent manner, and the IC 50 value ranged from 4.50 ± 0.06 to 10.60 ± 0.18 μg/ ml. YE showed an IC 50 value of 4.50 ± 0.06 μg/ml, which was the best among other extracts with 66.97% DPPH radical scavenging activity at 6.4 μg/ml. Ascorbic acid was used as positive control, which had the IC 50 value of 1.27 ± 0.01 μg/ml. In addition, water extract of yellow onion demonstrated potential DPPH scavenging activity with an IC 50 value of 6.77 ± 1.27 μg/ml, which was also better than the both extracts of red onion. Further, the IC 50 values of RE and RW were found to be 10.60 ± 0.18 μg/ml and 9.86 ± 1.40 μg/ml.
In addition, water extracts showed IC 50 value of 12.03 ± 0.23 and 29.04 ± 0.11 μg/ml for yellow and red onion cultivars, respectively.

| Inhibitory activities of onion extracts on protein tyrosine phosphatase 1B
The bioactivity of extracts on PTP1B inhibition was examined in vitro using pNPP substrate. The results of our study showed effective inhibition of PTP1B by the EtOH and water extracts of both red and yellow cultivars of onion, at concentrations of 0.4-10 μg/ml. The water extracts showed the highest activity, and at concentrations of 0.4, 2, and 10 μg/ml, the inhibition rates were in a range of 51.37%-98.34% (Figure 2a). In addition, YW with an IC 50 of 0.30 ± 0.08 μg/ ml was 10 times more potent than the positive control ursolic acid (3.40 ± 0.34 μg/ml) and 3 times more potent than the YE extract (0.86 ± 0.04 μg/ml). Further, RW also showed good inhibitory activity with an IC 50 of 0.33 ± 0.01 μg/ml, which was similar to the YW, but had higher activity than the YW at lower concentrations (0.4 and 2 μg/ml).

| Inhibitory activities of onion extracts on αglucosidase
All extracts significantly inhibited the α-glucosidase in a concentration-dependent manner (Figure 2b).

| Evaluation of cytotoxicity in HepG2 cells
In order to find out the non toxic concentration in HepG2 cells, we evaluated the cell viability using an MTT assay. As shown in Figure 4, water extract of both onion cultivars (RW and YW) showed no toxicity up to 100 μg/ml concentration. However, the 70% EtOH extract at 100 μg/ml showed 20% reduction in cell viability compared to normal control group.

| Glucose uptake potentials in insulin-resistant HepG2 cells
Considering the potent activity of extracts against PTP1B and wondering whether these extracts have ability to enhance glucose uptake, a 2-NBDG uptake assay was performed in insulin-resistant HepG2 cells. As shown in Figure 5, both solvent extracts of each type of cultivar showed a concentration-dependent glucose uptake activity. However, when compared to the water with ethanol extracts of each onion cultivar, water extract of both cultivars showed potent activity. The percentage of glucose uptake at 1.25 μg/ml was around 70% for 70% EtOH extract of both cultivars; however, at the same concentration, water extract showed about 90% uptake.

| PTP1B expression levels in insulin-resistant HepG2 cells
Protein tyrosine phosphatase 1B (PTP1B) regulates insulin signaling negatively, and its increased activity and expression are implicated in the pathogenesis of insulin resistance. In order to confirm whether HepG2 cells were treated with the selective concentrations of respective extracts. As shown in Figure 6, treatment of insulin-resistant HepG2 cells with onion extracts decreased the expression levels of PTP1B, which was in accordance with their glucose uptake potential.

| D ISCUSS I ON
Onions are among the most highly consumed vegetables worldwide in which flavonoids and the phenolic acids are the predominating ones. It was reported that polyphenolic compounds, including flavonoids, are safe and non toxic antioxidants. High dietary intake of natural phenolics has a strong correlation with longer life expectancy; reduced risk of developing some chronic disease, cancer, diabetes, and obesity; improved endothelial function; and blood pressure maintenance as suggested by various studies (Halliwell, 2007;Hodgson & Croft, 2006;Yan & Asmah, 2010).
In this study, we compared the 70% ethanol and water extracts of red and yellow onion peel cultivars for the TFC and TPC and found out that the level of TFC and TPC was higher in yellow onion F I G U R E 6 Effects of 70% EtOH and water extracts from onion on protein tyrosine phosphatase 1B (PTP1B) expression level in insulinresistant HepG2 cells. Western blotting was performed, and protein band intensities were quantified by densitometric analysis. Upper panels display representative blots. Equal protein loading was ensured and normalized against β-actin levels. Values are the mean ± SD of three independent experiments; a p < 0.001 indicates significant differences from the control group; b p < 0.01 and c p < 0.001 indicate significant differences from the 10 −6 M insulin-treated control group cultivars than red onion in both 70% ethanol and water extracts. The data comply with the result suggested by the Cheng et al. (2013) and are contrary to the study undertaken by Albishi, John, Al-Khalifa, and Shahidi (2013), Prakash, Singh, and Upadhyay (2007), and Shon, Choi, Kahng, Nam, and Sung (2004) on the content of TFC and TPC among cultivars. These authors used 50% methanol; a mixture of methanol, acetone, and water (7:7:6 v/v/v); and ethyl acetate for the extraction. However, we used 70% ethanol and water for extraction.
Ethanol was suggested as the best extraction solvent for almond hull extraction (Pinelo, Rubilar, Sineiro, & Nunez, 2004). A comparative study by Jung, Seog, Choi, Park, and Cho (2006) on TPC and TFC on different solvent extracts of wild ginseng leaves showed higher content in ethanol extract than methanol and water. Further, Patil, Pike, and Hamilton (1995) showed that the onion contents differ greatly with location, growing stage, and soil type among 75 cultivars grown in Texas.
Antioxidant assay methods are based on different mechanisms, so single method to evaluate the antioxidant activity is not appropriate (Prior, Wu, & Schaich, 2005). In our study, we showed the antioxidant activity of onion in red and yellow onion cultivars in 70% ethanol and water extracts via DPPH radical and ABTS radical scavenging activity. These all extracts showed good activity against them. However, the YE extract showed relatively higher activity among others in both DPPH radical scavenging activity and ABTS radical scavenging activity with the IC 50 value of 4.50 ± 0.06 and 6.64 ± 0.03 μg/mL, respectively. This may be due to the high level of TPC and TFC as suggested by Abdennacer, Karim, Nesrine, Mouna, and Mohamed (2015) and Pietta, Simonetti, and Mauri (1998). Yellow onion water extract had also shown greater antioxidant activity via DPPH radical scavenging activity and ABTS radical scavenging activity in accordance with the positive control. These data suggest that the peel can be used as natural antioxidants similar to the tea as suggested by Suzuki, Pervin, Goto, Isemura, and Nakamura (2016). The onion extracts had phenolic hydroxyl groups in the structure, and they have been recognized to function as electron or hydrogen donors (Shahidi, Janitha, & Wanasundara, 1992). According to Santas, Carbo, Gordon, and Almajano (2008) and Nuutila, Puupponen-Pimiä, Aarni, and Oksman-Caldentey (2003), flavonoids are the main compounds responsible for the antioxidant activity of onion peel.
Protein tyrosine phosphatase 1B (PTP1B) is an intracellular PTP localized on endoplasmic reticulum with its phosphatase domain oriented toward the cytoplasm and is expressed ubiquitously including in the classical insulin target tissues such as liver, muscle, and fat. Overexpression of PTP1B has been shown to inhibit signaling events both proximal and distal to the insulin receptor. Binding of insulin to insulin receptor leads to autophosphorylation of protein kinase. However, PTP1B interacts and removes tyrosine phosphates from insulin receptor. Further, PTP1B has the ability to dephosphorylate insulin receptor substrate proteins, thus attenuating and potentially terminating insulin signaling transduction (Zhang & Lee, 2003). It is reported that PTP1B-knockout mice exhibit high level of insulin sensitivity and are also resistant to high-fat diet-induced obesity. These specific results suggest that PTP1B plays a decisive role in the T2DM. Therefore, particular PTP1B inhibitors may have therapeutic benefits to T2DM (Fukuda et al., 2010). Over activity of PTP1B leads to the pathogenesis of insulin resistance in the obese patient. So the main strategy of T2DM is the inhibition of PTP1B (Johnson, Ermolieff, & Jirousek, 2002;Klaman et al., 2000;Koren & Fantus, 2007). In this study, we compared the PTP1B inhibitory activity of RE, RW, YE, and YW extracts. All the extracts showed high inhibitory activity, YW being the best. YW extract showed the 10 times more potent activity than the positive control ursolic acid.
Water extract had high activity than 70% ethanol extract against PTP1B. This recommends that water is the more appropriate solvent for extraction of compounds with PTP1B inhibitory activity from onion. Furthermore, in insulin-resistant HepG2 cell model, these extracts decreased the expression level of PTP1B in a concentrationdependent manner and enhanced the insulin-stimulated glucose uptake. From this result, we can speculate that the onion extract contains insulin sensitizers, which could be the reported compounds like quercetin and its derivatives along with other components that are yet to be reported. Owing to higher toxicity of ethanol extracts of both onion cultivars compared to water extract in our MTT assay and higher activity of water extract, overall results suggest that the water extract of yellow and red onion peels can be used in the prevention and management of T2DM. In addition, less toxicity of water extract might be attributed to the presence of highly glycosylated compounds as glycosylation leads to high polarity and reduced toxicity. So it is worth to characterize the specific novel components in the water extract that solely enhanced the glucose uptake by increasing the sensitivity of HepG2 cells toward insulin in the present study.
α-Glucosidase is secreted in the epithelium of small intestine, which acts as a key enzyme for carbohydrate digestion (converts disaccharides to monosaccharides). It has been recognized as a therapeutic target for the alleviation of postprandial hyperglycemia (Kim, Jeong, Wang, Lee, & Rhee, 2005). Thus, inhibitors can hinder the absorption of dietary carbohydrates followed by postprandial hyperglycemia suppression and could be powerful apparatus in easing hyperglycemia, and thus valuable for T2DM patients (Watanabe, Kawabata, Kurihara, & Niki, 1997). α-Glucosidase inhibitors are commonly used as oral hypoglycemic agents. But long-term use may cause flatulence, vomiting, and diarrhea (Hanefeld, 1998). There are numerous investigations regarding the discovery of α-glucosidase inhibitors from natural products having a lower side effect (Fujita, Yamagami, & Ohshima, 2001;Gholamhoseinian & Fallah, 2009;Shim et al., 2003;Wang, Du, & Song, 2010). In Korea, guava leaf extract and Touchi-extract have been approved as authorized health functional foods to improve postprandial hyperglycemia and in Japan, as Foods for Specified Health Use (FOSHU) (Kang et al., 2010).
In this comparative study, we evaluated the α-glucosidase inhibitory activity of RE, RW, YE and YW. All extracts showed the higher inhibitory activity in comparison with positive control (acarbose).
Among them, YE extract showed highest inhibitory activity against α-glucosidase, which was 20 times better than the positive control.
The second most active extract was YW. It was 12 times more potent than the positive control acarbose. The main target of diabetic therapy is to control fasting and postprandial hyperglycemia to maintain the blood glucose level. In comparison with fasting hyperglycemia, postprandial hyperglycemia is more strongly correlated with cardiovascular morbidity and mortality (Kang et al., 2010 To sum up, the comparative inhibitory activities of different extracts can be explained by their compositional differences. Phenolic and flavonoids were expected to be present in larger amount in the 70% ethanol extracts than the water extract, which is confirmed by our results. While comparing the antioxidant activity of onion peel between the different extracts, we found that the 70% ethanol extract had higher antioxidant activity than the water extracts. Additionally, Lee et al. (2014) reported the similar results while comparing the antioxidant activities of onion. Further, 70% ethanol extract showed better inhibitory activity on α-glucosidase and AGEs than the water extract on both cultivars. This may be due to the presence of high levels of TPC and TFC like quercetin along with other bioactive compounds as suggested by Kim et al. (2011).
Conversely, water extracts showed higher PTP1B inhibitory activity than the 70% ethanol extract. These data suggest that there are other more polar active compounds that are responsible for the specific inhibition of PTP1B activity. Due to the various side effects with the synthetic inhibitor, the current therapeutic effort has very limited success (Peyroux & Sternberg, 2006). Nutritional or herbal interventions based on the plant with high phenolic content represent a therapeutic approach with reduced risk of adverse effect as well as side effect and increase the patient compliance (Harris et al., 2011).

| CON CLUS ION
Overall, results indicate that the extract from peel of both red and yellow onions possesses a significant in vitro antioxidant and anti diabetic potential. The inhibitory activities of onion peel extract differed among red and yellow cultivars. At 2 μg/ml concentration, red onion peel extract displayed potent PTP1B inhibitory activity compared to yellow peel extract, while yellow onion had better αglucosidase inhibition. From this result, it may be concluded that red onion peel is superior in either quality or quantity of PTP1B inhibitors and/or insulin sensitizers to yellow onion peel. Similarly, among 70% ethanol and water as extracting solvents, 70% ethanol might be an appropriate for obtaining natural antioxidants. However, detail evaluation should be required to investigate the nature and amount of compounds in onion peel extract along with thorough in vivo studies.

| LI M ITATI O N S
The present comparative study was conducted on extracts of only two onion cultivars (red and yellow) using two extracting solvents (70% ethanol and water). This is an in vitro study. Further in vivo study must be conducted to confirm our findings.

This work was supported by Research Grant of Pukyong National
University (Granted in 2016).

CO N FLI C T O F I NTE R E S T
The authors declare that there are no conflict of interests.

E TH I C A L S TATEM ENTS
This study does not involve any human or animal testing.